Endothelial Nitric Oxide Synthase (eNOS) Gene Polymorphisms and Markers of Hemolysis, Inflammation and Endothelial Dysfunction in Brazilian Sickle Cell Anemia Patients.

The impaired bioavailability of endogenous nitric oxide (NO) in sickle cell anemia (SCA) may be influenced by polymorphisms in the endothelial nitric oxide synthase gene (eNOS). We compared allelic/genotypic frequencies of the eNOS polymorphisms T-786C, VNTR4a/b and G894T between 89 adult SCA patients and 100 healthy controls, and investigated the relationship between these SNPs and markers of hemolysis [lactate dehydrogenase (LDH), indirect bilirubin (IB) and reticulocyte counts], inflammation [interleukins IL-1ß, IL-6, IL-8, Tumor Necrosis Factor (TNF-α) and C-reactive protein (CRP)] and endothelial dysfunction (ED) [soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble L-selectin (sL-selectin), von Willebrand Factor (vWF) antigen and D-dimers] in the patients. The frequencies of the mutant -786C allele and -786C/C genotype were significantly higher in patients (p = 0.02 and p = 0.04, respectively) but not significantly correlated with the markers. For VNTR4a/b and G894T, the allelic/genotypic frequencies did not statistically differ between patient and control groups. Patients carrying the 4a allele and those with the 894G/G genotype showed a significant decrease in IB (p = 0.02 and p = 0.04, respectively), and only patients with the 4a allele exhibited reduced IL-1ß (p = 0.01). The correlation profiles between markers of inflammation and ED significantly differed between patients carrying the mutant alleles and those with wild-type genotypes. This appears to be the first report on the relationship between eNOS gene polymorphisms and markers of hemolysis, inflammation and ED in Brazilian SCA patients. Our results indicate that the SNPs analyzed may influence the phenotypic variability of these patients.

Understanding the molecular basis of the high oxygen affinity variant human hemoglobin Coimbra.

Human hemoglobin (Hb) Coimbra (ßAsp99Glu) is one of the seven ßAsp99 Hb variants described to date. All ßAsp99 substitutions result in increased affinity for O2 and decreased heme-heme cooperativity and their carriers are clinically characterized by erythrocytocis, caused by tissue hypoxia. Since ßAsp99 plays an important role in the allosteric α1ß2 interface and the mutation in Hb Coimbra only represents the insertion of a CH2 group in this interface, the present study of Hb Coimbra is important for a better understanding of the global impact of small modifications in this allosteric interface. We carried out functional, kinetic and dynamic characterization of this hemoglobin, focusing on the interpretation of these results in the context of a growth of the position 99 side chain length in the α1ß2 interface. Oxygen affinity was evaluated by measuring p50 values in distinct pHs (Bohr effect), and the heme-heme cooperativity was analyzed by determining the Hill coefficient (n), in addition to the effect of the allosteric effectors inositol hexaphosphate (IHP) and 2,3-bisphosphoglyceric acid (2,3-BPG). Computer simulations revealed a stabilization of the R state in the Coimbra variant with respect to the wild type, and consistently, the T-to-R quaternary transition was observed on the nanosecond time scale of classical molecular dynamics simulations.

A new ß°-thalassemia frameshift mutation [ß 48 (-T)] in a Uruguayan family.

We describe here a new frameshift mutation of ß-thalassemia in a Uruguayan family with Italian ancestry [ß48 (-T); HBB:c.146delT]. This frameshift results in formation of premature stop codon (TGA) 40 bp downstream and in a short unstable product that is degraded in the cell.

Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis.

Phosphatidylinositol phosphate kinases (PIPKs) are enzymes that participate in diverse intracellular signaling pathways. They are classified into 3 functionally distinct subfamilies - PIPKI (α, ß, γ), PIPKII (α, ß, γ), and PIPKIII - located in various subcellular compartments. Recently, the PIPKIIα and ß-globin genes were found to be overexpressed in reticulocytes from 2 siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIa and the production of globins. The main aim of this study was to determine the expression profiles of PIPK genes in healthy individuals during in vitro erythropoiesis using quantitative real-time polymerase chain reaction and to compare these profiles with profiles of globin genes. Our results showed that expression of all PIPKs increases as the cells differentiate, coinciding with the expression profiles of globins. Analysis of the effects of globins on PIPK genes revealed that they varied significantly between the globins, the most noticeable being the effect of α-globin on PIPKIIα (P < 0.0001) and γ-globin on PIPKIIγ (P < 0.0001). The relationship between the expression of PIPKs and globin genes was statistically significant, particularly between PIPKIIα and α-globin (P = 0.0002) and PIPKIIγ and ß-globin (P < 0.0001). Linear correlation analysis revealed a strong relationship between PIPKIIα and α-globin genes. This study is the first to establish the expression profiles of PIPK genes during in vitro erythropoiesis in healthy individuals and suggests a parallel between the expression of PIPK and globin genes, reinforcing the hypothesis that they may be related.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population.

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression.

The alpha-MRE is the major regulatory element responsible for the expression of human alpha-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the alpha-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the alpha-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different alpha-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type alpha-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G-->A polymorphism at positions +130, +199, and +209, separately, were also tested. The different alpha-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C-->A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of alpha-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Regulation of human alpha-globin gene expression and alpha-thalassemia.

Hemoglobin and globin genes are important models for studying protein and gene structure, function and regulation. We reviewed the main aspects of regulation of human alpha-globin synthesis, encoded by two adjacent genes (alpha(2) and alpha(1)) clustered on chromosome 16. Their expression is controlled mainly by a regulatory element located 40 kb upstream on the same chromosome, the alpha-major regulatory element, whose activity is restricted to a core fragment of 350 bp, within which several regulatory protein binding sites have been found. Natural deletions involving alpha-major regulatory element constitute a particular category of alpha-thalassemia determinants in which the alpha-globin genes are physically intact but functionally inactive.

Hb Indianapolis [beta112 (G14) Cys-->Arg] as the probable cause of moderate hemolytic anemia and renal damage in a Brazilian patient.

Hemoglobin (Hb) Indianapolis [beta112 (G14) Cys-->Arg] is a rare and slightly unstable beta-globin variant. All carriers described to date were clinically normal with only mild reticulocytosis. We report here a case of a Brazilian patient in whom hemolytic anemia and acute renal failure were probably caused by the presence of this variant.

Hb Florida: a novel elongated C-terminal beta-globin variant causing dominant beta-thalassemia phenotype.

We report here a new frameshift mutation in exon 3 of the beta-globin gene, a single nucleotide deletion (-C) in between codons 140/141 (GCC/CTG-->GCC/TG), found in an 8-year-old Argentinean girl with clinical picture of thalassemia intermedia. It leads to a beta-chain that is elongated to 156 amino acids [(141)Trp-Pro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-Leu-Leu-Ser-Asn-Phe-(156)Tyr-COOH]. The resulting hemoglobin, which we named Hb Florida, was not detected in peripheral blood; however, erythroid hyperplasia and dyserythropoiesis with large inclusion bodies on methyl violet staining were observed in bone marrow, suggesting that this is a hyperunstable variant producing a dominant beta-thalassemia phenotype, since the other beta-allele was completely normal.

Reticulocyte evaluation in alpha(+)-thalassemia.

Although it is almost certain that alpha(+)-thalassemia protects against malaria, the mechanisms for that are still unknown. It has been suggested that an increased number of young circulating red blood cells in alpha(+)-thalassemic children, as a result of some degree of ineffective erythropoiesis, could be related to the high frequencies of the alpha(+)-thalassemic allele in malaria endemic areas. Reticulocyte evaluation in this condition, however, has been poorly performed so far. Our objective was to determine the reticulocyte number and maturation degree, in addition to the soluble transferrin receptor and serum erythropoietin levels, in alpha(+)-thalassemia heterozygotes, comparing them with normal alpha-genotype controls. One hundred twenty-one alpha(+)-thalassemia carriers (-alpha(3.7)/alphaalpha) and 249 controls (alphaalpha/alphaalpha), all of them with normal serum ferritin levels, were subclassified according to age (1-5, 6-10, 11-15, 16-20, and over 20 years old). Reticulocyte analyzes were carried out by flow cytometry and sTfR and s-Epo levels determined by immunonephelometry and chemiluminescence, respectively. The comparisons did not show any significant difference between thalassemics and controls regarding the reticulocyte parameters [percentages and absolute values, P = 0.2643 and 0.5421; high, medium, and low maturation degree, P = 0.2579, 0.2196, and 0.4192; RET maturity index (RMI), P = 0.2471, respectively], as well as the s-Epo levels (P = 0.5711). The sTfR concentrations were higher in the thalassemic group (P = 0.0001), but statistical significance was due only to the 1-5 and over 20 subgroups (P = 0.0082 and 0.0436, respectively). The results found here are compatible with a compensated erythropoiesis and do not confirm the hypothesis mentioned above.

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystemÖ and GenePhorÖ, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystemÖ and 20 percent gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.